Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection.
Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection.
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发表时间：
1994-04
期刊：
影响因子：
2.7
通讯作者：
H. Chen;R. S. Nelson;Sherwood Jl
中科院分区：
文献类型：
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作者：
H. Chen;R. S. Nelson;Sherwood Jl
冻融转化为直接转化根癌农杆菌提供了一种简便、快速的方法。用4~9个大小、插入片段大小不同的质粒转化LBA4404、GV3850和EHA101菌株的根癌农杆菌感受态细胞，在某些情况下对抗生素敏感。与干冰/乙醇相比，用液氮冷冻细胞每微克DNA的转化菌落数增加了三到四倍。在液氮中冷冻细胞，然后在培养前将转化细胞在低浓度的适当抗生素中孵育，与在培养前没有在低浓度抗生素中孵育的干冰/乙醇中冷冻细胞相比，获得的集落数增加了9倍。来自质粒的预期大小的限制性片段表明，程序修改没有在插入区域引起明显的重组。
Freeze-thaw transformation provides a simple and rapid method to transform Agrobacterium tumefaciens directly with plasmid DNA. Competent A. tumefaciens cells of strains LBA4404, GV3850 and EHA101 were transformed with four to nine plasmids differing in size, size of insert and in some cases sensitivity to antibiotics. A threefold to fourfold increase in transformed colonies per microgram of DNA was obtained by freezing cells with liquid nitrogen vs. dry ice/ethanol. Freezing cells in liquid nitrogen followed by incubation of transformed cells in a low concentration of appropriate antibiotics prior to plating resulted in a ninefold increase in colonies obtained compared with the procedure of freezing cells in dry ice/ethanol without the incubation period in the low concentration of antibiotics prior to plating. Restriction fragments of the expected sizes from the plasmids indicated that the procedural modifications did not cause apparent recombinations in the region of the inserts.